Method of determining ovulation time in urine and fertility in females

ABSTRACT

THE PRESENCE OF ANTERIOR PITUITARY HORMONE IN MAMMALIAN URINE, BLOOD, SERUM OR PLASMA IS DETERMINED BY MIXING WITH THE URINE, BLOOD, SERUM OR PLASMA A TEST QUANTITY OF A STABLE SOLUTION OF 2-4, DINITROPHENYLHYDRAZINE AND AN AQUEOUS SOLUTION OF AN ALKALI METAL HYDROXIDE AND THEN COMPARING THE COLOR OF THE SOLUTION WITH A CONTROL COLOR.

Un ted Sta es Pa e 3,813,222 Patented May 28, 1974 METHOD OF DETERMININGOVULATION TIME IN URINE AND FERTILITY IN FEMALES I. R. La Vietes, BelleHarbor, N.Y., assignor to La Mar Laboratories, Inc. Filed Oct. 14, 1971,Ser. No. 189,249 Int. Cl. Gtlln 31/22, 33/16; G07c 87/48 US. Cl. 23-230B Claims ABSTRACT OF THE DISCLOSURE The presence of anterior pituitaryhormone in mammalian urine, blood, serum or plasma is determined bymixing with the urine, blood, serum or plasma a test quantity of astable solution of 2-4, dinitrophenylhydrazine and an aqueous solutionof an alkali metal hydroxide and then comparing the color of thesolution with a control color.

The present invention relates to the preparation of a reagent that isused to detect the presence of small amounts (20-50 micrograms) ofanterior pituitary hormone (estrogen) in mammalian urine, blood, serumor plasma.

Another object of the invention is the utilization of said reagent todetermine the time, during a female menstrual or oestrus cycle, whenovulation takes place.

Determination of estrogen levels is of interest to physicians inelucidating atypical hormonal patterns in order to provide propertreatment for a variety of disease conditions such as abortion,hypergonadism and hypogonadism. Determination of ovulation time andcommensurately, whether, ovulation takes'place at all is an importantphysiological consideration. Information about this function is ofinterest to women who find it diflicult to conceive because of atypicalmenstrual cycles; to individuals who wish to regulate family size butbecause of a variety of considerations do not choose to usecontraceptives; to the physician in assisting him in the diagnosis ofseveral abnormal conditions such as abortion and sterility, and to theveterinarian in determining times of fecundity in household pets andanimal stock.

Heretofore, the determination of ovulation time was a hit and missaffair which employed, in the case of humans, the taking of vaginaltemperatures over a period of months, testing the vaginal mucosa forglucose which is difficult to perform and inaccurate or determiningestrogen levels by means of a long and costly chromatographic procedure.

This product covered by this invention was required to fulfill twocriteria; sensitivity to the low levels of estrogen present in normalnon-gravid females and stability over a range of room temperatures (40degrees-90 degrees F.) over long period of time (6-9 months).

The compound 2,4 dinitrophenylhydrazine (DNPH) is characterized by thepresence of both hydrophilic and hydrophobic moieties which accounts forits well known limited solubility in almost all solvents and its limitedstability once in solution. To'provide a means for increasing thesolubility of the compound to the point where it would be effective as areagent, solution was effected in.

hot hydrochloric acid. Similar results with varying degrees ofsolubility can also be ,obtained with other mineral acids, water andconcentrated or dilute alcohols. To provide a means of stabilizing thecompound once in solution a polyhydric alcohol such as polyethyleneglycol was added. Similar results with varying degrees of stability canalso be obtained with other polyhydric alcohols such as propylene glycoland glycerin.

The reagent can be typically prepared in the following manner:

A stock solution is made by dissolving 1 gram of DNPH and 0.1 gm. ofmethyl red in 175 ml. of concentrated hydrochloric acid and 50 ml. ofpolyethylene glycol with the aid of heat. The solution is then allowedto cool. Varying amounts of polyethylene glycol are then added to obtaina standardized solution. The solution of DNPH and the polyhydric alcoholshould be 4.5-6.5 N, preferably about 5.5 N. No fixed amount can be seton the volume of diluent (polyethylene glycol) to be added forcommercially prepared DNPH varies considerably from lot to lot, withrespect to strength and purity. Standardization therefore, takes placeby colorimetrically matching the solution against known standards.

The methyl red serves as a pH indicator and to intensify the colorsobtained when the reaction has gone to completion. Other indicators of asimilar type may be used such as methyl orange or methyl purple, or theindicator itself may be eliminated from the reagent completely.

The reagent described above (Reagent A) is used in conjunction with asecond reagent (Reagent B) which is an aqueous solution of an alkalimetal hydroxidesuch as potassium or sodium hydroxide. The second reagentsolution should be 59.7 N, preferably 6.4 N. It is made in accordancewith known procedures and of sufficient strength to give a final pH of9-115 when used in the described procedure.

Urine was collected on a daily basis over a period of several months,from women who had a history of regular menstrual cycles. These womenwere instructed to take their basal temperature daily so that testresults could be correlated through a corroborative method. The urinewas tested daily by the described process.

There are provided 2 like vessels, as for example tubes or vials ofapproximately diameter and 2" height, these vessels can be calibratedfor convenience, as for example at the 7 ml. level or a measuring devicesuch as a. pipette can be used to dispense the reagents and/or urine.The vessels can be marked appropriately to aid in distinguishing betweenthem. A specific volume of Reagent A is added to one of the vessels(Tube 1) to which is also added urine to the volume mark on the vesselor by means of a calibrated dropping device. The contents are shaken toeifect mixture and the vessel is set aside. A predetermined time periodsuch as seconds is allowed to elapse after which a specific volume ofReagent B is added to Tube 1 and contents mixed. Tube 1 is set aside.

A specific volume of Reagent A is added to he remaining vessel (Tube 2)to which is also added a specific volume of urine from hte urine sampleas used in Tube 1 by means of a calibrated dispensing device or to apre-calibrated level marked on the vessel. A specific amount of ReagentB is added to Tube 2 immediately. The vessel is shaken to mix thecontents and set aside. Tube 2 is meant to be a control against whichthe colors resulting from the chemical reaction in Tube 1 is compared.Tube 2 contains all the components found in Tube 1 except that thereaction has not gone to completion. The pH of the final solution inboth tubes should be about 9-1 1.5.

After a time period, as for example, 3 minutes, Tube 1 and Tube 2 arecompared visually or optically by means of a photometer. If the color ofthe liquid in Tube 1 is the same color or slightly lighter or darkerthan the color of the liquid in Tube 2, the reaction indicates thatovulation is taking place or has taken place within the last 24 hours,or will take place within the next 24 hours. If the color of the liquidin Tube 1 is much darker and in marked contrast to the liquid in Tube 2,then the reaction indicates that ovulation is not taking place.

A time element is involved with the test. The sensitivity of the testcan be decreased by increasing the time element between the addition ofReagent A and Reagent B. This is an advantage as it gives an alternativemethod of increasing or decreasing the sensitivity of the test. The

test may be performed in a single vessel (Tube 1) without recourse tothe use of a control sample. The color of the liquid in said Tube 1 maybe compared to standards prepared previously that have been made on orin paper, glass, plastic and other like material or to previouslyprepared liquid samples of known concentration. The final colors mayalso be described verbally so that no standard or control need beemployed as a means of comparison there being a marked difference in thecolor and lightnesses of the liquid resulting from a reaction indicatingovulation and a reaction indicating no ovulation.

What I claim is:

1. A method for detecting the presence of anterior pituitary hormone inurine, blood, serum or plasma of non-pregnant female mammals whichcomprises, in the following sequence:

Placing the urine, blood, serum or plasma in a test container;

Adding thereto a specific amount of a solution of 2,4-dinitrophenylhydrazine in a solvent, the solution having been stabilizedby the addition of a polyhydric alcohol;

Allowing the mixture to stand for about 90 seconds thereby forming acolor;

Adding a specific amount of an aqueous solution of alkali metalhydroxide whereby a fin-al solution having a pH of 9-1 1.5 is formed;and

Measuring the intensity of color of the final solution after a secondtime period, thereby determining whether ovulation has taken place.

2. A process for the preparation of a stable solution of2,4-dinitrophenylhydrazine which comprises dissolving the2,4-dinitrophenylhydrazine in a solvent and adding a polyhydric alcoholthereto, said polyhydric alcohol being a stabilizer.

3. The process as recited in claim 2 wherein the solvent is water.

4. The process as recited in claim 2 wherein the polyhydric alcohol ispolyethylene glycol.

5. The process as recited in claim 2 in which the solvent is selectedfrom the group consisting of alcohol and a mixture of water and alcohol.

6. The process of claim 2 in which the polyhydn'c alcohol is selectedfrom the group consisting of propylene glycol and glycerine.

7. The process as recited in claim 2 wherein the solvent is a mineralacid.

8. The process as recited in claim 7 wherein the mineral acid ishydrochloric acid.

9. The process as recited in claim 2 wherein a pH indicator is added tothe stable solution.

10. The process of claim 9 in which the pH indicator is selected fromthe group consisting of methyl red, methyl orange, and methyl purple.

References Cited UNITED STATES PATENTS 3,434,801 3/ 1969 Scherr 252408 X3,436,186 4/1969 McSweeney et al. 23230 B 3,226,196 12/1965 La Vietes23230 B OTHER REFERENCES L. Kahlenberg, Science 46, No. 1578, 344(1925). Machbeth et al., Chem. Abstr. 29, 750 (1935). Roduta et al.,Chem. Abstr. 31, 98 5 (1937). Reich et al., Chem. Abstr. 50, 395 c(1956).

L. Legradi, Chem. Abstr. 63, 12296 g (1965).

ROBERT M. REESE, Primary Examiner U.S. Cl. X.R.

